Pain affects over 100 million Americans annually. Opioids are the primary drugs that can treat pain. However, these drugs produce severe and unwanted side effects, such as respiratory depression, nausea, constipation and tolerance development (Berde et al, 2008); in addition, they are highly addictive (Koob et al, 1998). There is clearly a large, unmet need for drugs that can produce analgesia with limited or no side effects. The peptide neuromedin U (NMU) is believed to be an integral component of pain pathways (Yu et al, 2003). NMU acts through two distinct receptors - the neuromedin U receptor-1 (NMUR1) and 2 (NMUR2) (Brighton et al, 2004). NMUR1 is expressed predominantly in the periphery, with the highest levels in the gastrointestinal tract (Hedrick et al, 2000), while NMUR2 is mainly expressed in the central nervous system (CNS) (Shan et al, 2000) including regions of the brain implicated in pain (Torres R, 2007). We have shown that NMUR2 knockout mice have reduced pain sensitivity, whereas other physiological activity and responses appear normal. These findings make NMUR2 an attractive drug target and suggest that NMUR2 antagonists may be useful analgesics with potentially limited side effects. In the Phase I grant we proposed to validate NMUR2 antagonists as novel analgesics. To this end, we a) identified several structurally diverse classes of NMUR2 antagonists through screening of small-molecule chemical libraries and b) demonstrated in vivo efficacy in nociceptive animal models with one of the antagonists that was resynthesized following its identification using the screening library. With the successful identification of several classes of molecules from the earlier studies, we now propose in this Phase II grant to begin the optimization and preclinical development of validated screening hits. A primary goal will be to synthesize and test compounds in order to produce optimized molecules having CNS drug-like attributes and the desired pharmacological effect. This will be accomplished using a traditional drug medicinal chemistry approach to define structure-activity relationships (SAR) and ultimately construct molecules that have the necessary CNS characteristics. Our plan is to select screening hits from two diverse chemical classes. Targeted compounds will first be chemically synthesized and then tested for potency in a cell-based assay for NMUR2 antagonism and receptor selectivity. Next, the compounds will be subjected to a battery of in vitro absorption, distribution, metabolism and excretion (ADME) tests and compound profiling assays to optimize compound properties. The most promising compounds will advance to in vivo efficacy and pharmacokinetic testing to provide additional data for further compound optimization. The goal will be to identify multiple candidates that will then be poised for additional preclinical toxicology studies.